Angiogenesis is essential for normal physiological processes such as organ development and wound healing. It is also critical to pathological processes such as tumor growth, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. Therefore, blocking angiogenesis could be a powerful therapeutic intervention for treating diseases requiring formation of new blood vessels. However, the underlying molecular mechanism of intracellular signal transduction in this process remains largely unexplored. We recently generated Mekk3-knockout mice through homologous recombination and demonstrated that MEKK3, a Ser/Thr protein kinase belonging to the mitogen-activated protein kinase (MAPK) kinase gene family, is essential for angiogenesis. The long-term goals of this study are to elucidate the molecular mechanisms of angiogenesis regulated by MEKK3 signaling. Our SPECIFIC AIM 1 is to determine the function of MEKK3 in endothelial cells (ECs) during embryonic development by examining the morphology, proliferation and apoptosis of ECs in the E8.5 to E10 wild-type, Mekk3+/- and Mekk3-/- fetuses. We will also isolate embryonic ECs from the E9-E9.5 fetuses and establish EC lines by using polyoma middle T antigen to transform the primary cultures. In SPECIFIC AIM 2, we will determine whether MEKK3 is a specific upstream activator of JNK1/2, ERK1/2, p38 and ERK5 MAPKs in ECs by using in vitro kinase assays. We will determine whether the induction of these MAPKs is defective in Mekk3-/- embryos, purified ECs and EC lines. In addition, we will determine whether MEKK3 is specifically activated by angiogenic stimulation by using in vitro kinase, in-gel kinase and MEKK3 phosphorylation assays. Furthermore, we will isolate and clone MEKK3-associated proteins by coprecipitation, chromatography, and yeast two-hybrid screening. In Specific Aim 4, we plan to generate Mekk3 floxed ES cells and mice, and use these mice to create EC-specific Mekk3 knock out (Mekk3 (EC-null)) mice and Mekk3-deficient ECs with Cre recombinase. Function of MEKK3-signaling will be studied specifically in ECs using these mice. In addition to revealing the MEKK3-signaling in angiogenesis, the outcome from this study will provide conceptual and material resources for studying MEKK3 and its homologues in many other physiological and pathological processes. Most importantly, this study may discover novel molecular targets for therapeutic intervention for treating diseases requiring formation of new blood vessels like cancer.